Biological samples or materials are frequently examined microscopically. Particularly, appropriate objects can be examined for detection of structures with wide field optical elements, which show the object and/or a thin layer, ideally a plane, of said object on a resolving detector. Such imaging examination can be carried out, for example, with standard microscopy or fluorescence microscopy. The thin layer, for example, can be a fluorescent layer on a slide, such as a microscopic slide or the bottom of a titer plate, which contains immobilized cells, tissue sections, or DNA fields, preferably arranged in microarrays.
However, particularly for the examination of biological objects, quantitative fluorescence microscopy is also frequently used. Generally, quantitative fluorescence microscopy, by means of irradiation of a sample, particularly a thin layer of defined thickness, aims to induce fluorescence radiation, the intensity of which depends on the concentration of fluorescing substances in the sample to be measured. Measuring the intensity of fluorescence radiation allows for conclusions regarding the concentration of the fluorescing substances. Therefore, extreme image resolution is less important that a reliable detection of the radiation emanating solely from the thin layer.
As a result, instead of high-resolution microscopes, so-called spatially resolving fluorescence readers are used, which can be viewed as optimized microscopes for quantitative fluorescence microscopy. In particular, the objects and/or samples can be biochips, manufactured either photolithographically or by means of a spotter.
In order to receive the most precise reading for the intensity of the fluorescence radiation generated in the thin layer, two conditions must be observed. On one hand, the fluorescence radiation from the thin layer should be recorded as comprehensively as possible and quantitatively precise. On the other hand, any radiation, particularly fluorescence radiation, which does not originate from the thin layer, should be suppressed as much as possible, i.e., a sufficient in-depth selection should be achieved, recording only radiation in one layer around the focal plane. The optical radiation to be suppressed shall hereinafter be called artificial light, even though it does not necessarily have to be within the visual range of the optical spectrum.
At least the following sources can be considered sources of artificial light. For example, artificial light originates from reflections and stray light on surfaces, in glasses, e.g., due to entrapped air, intrinsic fluorescence of the used glasses, on frames or in fluorescence measurements from unsuppressed excitation light. Furthermore, artificial light can also originate from areas of the object or the sample, which are outside of the focal plane located preferably within the thin layer, for example, from fluorescing contaminations on the reverse side of the object slide or from an adjacent layer with a fluorescent liquid.
However, artificial light can also compromise the mapping of the object because it decreases or alters the contrast of the detected intensity distribution.
The use of confocal laser scanners provides a possibility for avoiding artificial light. With a confocal laser scanner, always only one small area of the sample of a few Fm2 is illuminated, and in addition, only this small area is viewed during detection. If executed consistently with the help of a sufficiently adjusted aperture, the artificial light will be suppressed from the start. However, compared to microscopes with wide field optical element, laser scanners exhibit a number of disadvantages. For example, excitation saturation and intense bleaching of fluorophores due to the high radiation intensity in the focal point may occur during fluorescence microscopy. Furthermore, there are significant limitations regarding the choice of wave length. Additional disadvantages are many movable components, high adjustment requirements as well as low quantum efficiency of the detector, as a rule a photomultiplier.
In order to avoid said disadvantages, methods are suggested which detect images under various illuminating patterns and calculate an image of the thin layer from the detected images.
For example, EP 972220 B1 describes a method, whereby three images of the object with the thin layer are detected, which are detected through illumination, focused on the thin layer, with spatially sinusoidal intensity profiles, which are phase-shifted by a third of a period against each other.
From the detected images, an image of the thin layer is calculated.
In DE 199 30 816 A1, a method and a device for in-depth selection of microscope images are described, whereby a one-dimensional periodic grid, e.g., a stripe grid, is used for illumination. In this case, at least n (n>2) CCD camera images are taken, whereby the structure of the illumination is shifted by 1/n each of the grid constants. Consequently, from at least three images a confocal section of the sample is calculated. This method is susceptible to artifacts when the grid does not produce sinusoidal illumination intensity on the sample.
WO 98/45745 A1 (DE 698 02 514 T2) describes an imaging system and method for microscopes, which provides structured illumination through superimposition of two coherent light beams. The method, similar to the aforementioned method, according to DE 199 30 816 A1, mainly aims to generate optical sections in various object planes analogously to a laser scanning microscope.
Both methods aim to receive a depth resolution of thick samples. Their purpose is to produce, with a wide field optical element, confocal sections of a thick sample, when compared to the depth of focus, or an object. In both cases, the calculations are relatively labor-intensive because trigonometric equations must be solved.
In the unpublished German patent application P 103 30 716.8, a device for the execution of a method for eliminating artificial light on images of heterogeneous, luminous or illuminated two-dimensional objects is described. It includes a radiation source with subsequent illuminating optical element, which homogenize the radiation, for homogenous illumination of a subsequent field stop plane, in which a structured field stop for creating an illumination structure, which is superimposed over an object or a sample, is arranged. Said illumination structure is mapped onto the sample through initial optical means, whereby said initial optical means may include an illumination tube, possibly a dichroic mirror, and a lens. Furthermore, secondary optical means for mapping the sample together with the superimposed illumination structure onto a resolving detector, particularly for optical radiation, are provided. Additionally, the arrangement contains means for adjustment, with which the illumination structure can be definably positioned in the object plane on the object or the sample. The detector is connected to an evaluation installation for determining and eliminating the artificial light. A structured bright field illumination with at least two different illumination patterns is used, whereby dark areas do not overlap. Subsequently, a dark frame and a light frame can be determined from respective images. Subtraction of the dark frame from the light frame leads to the resulting image.
The structured bright field illumination provided for this device, in which the object illumination and the mapping of the object together with the superimposed field stop structure are achieved with a single lens, the excitation light in the lens can cause the occurrence of artificial light, particularly through intrinsic fluorescence of the applied glasses.
Furthermore, the reverse side of an object, e.g., a biochip, is exposed to almost the same excitation intensity as the focal plane. Therefore, the fluorescence intensity, caused by contamination of the reverse side, can be correspondingly high and lead to errors in measurements. In order to avoid said disadvantages, it was, therefore, suggested to use a structured dark field illumination instead of the bright field illumination.
Both methods require execution of an interpolation between the non-illuminated areas in order to obtain a complete dark frame and/or artificial light image.
However, for the quantitative fluorescence microscopy, all aforementioned methods have the disadvantage that the precision of the concentration measurements of fluorescent material in the thin layer is still improvable, even though an in-depth selection is achievable and artificial light from the areas adjacent to the thin layer can be at least partially suppressed.